JPH04506457A - Production and use of human NM23 protein and antibodies thereto - Google Patents

Abstract

Human nm23 DNA and protein is disclosed as well as antibodies which recognize human nm23 protein. The DNA and antibodies may be used to detect nm23 in human tumors to predict the malignancy potential of such tumors.

Description

[Detailed description of the invention] Human nm23 protein and it Production and use of antibodies against Background of the invention The present invention relates to human nm23 protein and DNA encoding human nm23 protein. (or such a fragment or analog of NA), human nm23 protein antibodies that recognize these substances, as well as processes and production methods for producing and using such substances. relating to things.

Steeg et al,, Journal or the National Cancer Institute, 80:200-204, 191+8 Mouse nm23 gene and corresponding protein involved in tumor metastatic potential will be disclosed. The applicant has identified the human gene encoding the human nm23 protein, The present invention provides proteins and antibodies useful in predicting the invasion of human tumors. Main departure According to one aspect of the Ming Dynasty, DNA encoding human nm23 protein or such Fragments, analogs or derivatives of NA are provided.

According to another aspect of the invention, DNA encoding human nm23 protein or Cloning or production involving such fragments, analogs or derivatives of NA. A current vehicle is provided.

According to another aspect of the invention, a host; in particular human nm23 protein or such DNA encoding a fragment, analog or derivative of NA or such NA cells that have been genetically engineered to contain fragments, analogs or derivatives of; i.e. Such cells modified to contain human nm23 DNA are provided.

According to yet another aspect of the invention, human nm23 protein is provided.

According to yet another aspect of the invention, an antibody that recognizes human nm23 protein is provided. It will be done.

According to still another aspect of the invention, the manipulation for using said DNA and the metastatic potential of a tumor are provided. Antibodies for predicting potency are provided.

The term "human nm23 gene or DNA" as used herein refers to the human nm2 The gene or DNA encoding the 3 protein or the human nm23 protein Such NA or Refers to an analogue, derivative or fragment of a gene. Therefore, the human nm23 gene The term refers to the gene or DNA shown in Figure 2 and the gene or DNA shown in Figure 3. , a fraction thereof or a fragment, derivative or analog of such a gene. Contains.

“Human nm23 protein ° is an amino acid sequence unique to human nm23 protein. and preferably an antibody recognized by human nm23 protein. The nm23 protein found in humans or its fragments and analogs also induces or a derivative. The term human nm23 protein is used in Figure 2 or Figure 3. Includes proteins encoded by the genes shown.

The term "antibody" as used herein refers to polyclonal and monoclonal antibodies. includes.

Is the term “nm23 antibody” induced in response to human nm23 protein? Or it means an antibody that recognizes human nm23 protein. human nm23 protein Antibodies recognizing the quality may or may not be induced in response to human nm23 protein. You don't have to.

Applicants herein present two proteins encoding two different and distinct nm23 proteins. discovered different and distinct human genes (DNA). The first gene is shown in Figure 2. and is referred to herein as nm23-Hl. The second gene here is nm23-H 2, and a portion of such a gene sequence is shown in FIG. In Figure 3 Base pair number 1 corresponds to base pair number 216 in FIG.

Applicants hereby identify two distinct proteins encoding two different nm23 proteins. However, the scope of the present invention is not limited to such specific genes.

Human nm23 DNA (RNA) detects and/or measures RNA or DNA It can be used as a diagnostic tool for For example, it would look like this: NA or RN A to detect mRNA expression in cancer cells and thereby determine the malignant potential of human tumors. May be used to help predict forces. The following methods are available: (1) RNA (“Northern”) plotting, RNA can be plotted using several standards. can be isolated from tumor samples by any of the following procedures: For example, Le A modification of the method of hrach (1) can be used. RNA was analyzed by denaturing gel electrophoresis. and transferred to nitrocellulose or other support matrix. nm23 mRNA can be isolated from radioactive or non-radioactive hives of nm23-H1 or nm23-H2. Can be detected by lid formation. The mRNA in the tumor varies depending on the strength of hybridization. It will be shown. For comparison, a control probe for mRNA with constant levels (eg, β-actin) to normalize the results. low level Detection of nm23-Hl or nm23-H2 in the tumor indicates a tumor with high malignant potential.

(2) Nuclease protection assay, RNA isolated from tumor samples is labeled nm23-Hl or It can be analyzed for the content of nm23-H2. nm23-Hl or nm2 Using all or part of the B-H2 nucleotide sequence, the plasmid can be linked to the corresponding mRNA. can be formed for the production of nucleic acid probes complementary to A. As an example of such a vector Then, vectors based on T7 or SP6 promoters for RNA probes, Or m13 for DNA probe production using oligonucleotide brining There is a fuage. Probes produced from such vectors contain excess probe completely hybridizes with RNA from tumor samples under conditions of . RN Ah S can be used to remove molar unhybridized RNA probes, or S 1 nuclease or other single-strand specific DNase is a non-hybridizing N Can be used to remove the A probe. These are then subjected to denaturing gel electrophoresis and Subjected to autoradiography. The intensity of the band corresponding to the protection probe is It is either the amount of nm23-Hl or nm23-Hl from the sample. Re If we also include nuclease protection experiments for mRNAs with unchanged bells, the results can be standardized. relatively low levels of nm23-Hl or nm23-Hl Detection of a tumor indicates a tumor with high malignant potential.

(2) In situ hybridization of nm23-Hl or nm2B-Hl in tumor sections The amount of nm23-Hl or Hl mRNA in individual cells of the tumor is analyzed. Probes complementary to nm23-Hl or H2 sequences were prepared as described above. can be manufactured and embedded by standard techniques (such as by the use of paraffin or hybridization with mRNA in thin sections of tumor samples (preserved at Wear. Non-hybridized probes can be removed with nucleases. hybrid Formation can be detected by autoradiography or other methods. of hybridization Intensity reflects the amount of nm23-Ml or Hl mRNA within tumor cells.

If tumor cells contain low levels of nm23, they are considered highly malignant. be exposed.

Human nm23 DNA (RNA) has this abnormality in NA in human cancer cells. may be used to help detect and thereby predict cancer invasion ( Abnormalities are seen in more aggressive cells). Such methods include: (1) DNA isolated from tumors was analyzed by plot hybridization to nm2 3-Hl or H2 gene abnormalities can be tested. From normal tissue and tumor tissue The isolated DNA was fragmented with restriction enzymes, subjected to gel electrophoresis, and nm23-Hl and H2 gene fragments are transferred to a loin or other support matrix. is the whole or part of the cDNA or the nm23-Hl or H2 gene. Detected by hybridization using probes containing other regions (Southern sample) lot operations).

The difference in hybridization patterns between DNA from normal or tumor cells is n m23- Indicates an abnormality related to the Hl or H2 gene.

(2) Confirmation of allelic deletion regarding nm23-Hl or H2 gene. each nm2 Restriction chain long winter type (RFLP) for the three genes can be confirmed by Southern blot operation. I can do it. RFLP is a genetic disorder in patients who are heterozygous for RFLP. It may also be used to identify individual alleles for a gene. - the patient's normal DNA from tumor cells and tumors D for either nm2B-Hl or Hl If NA indicates that there is an allelic deletion, such changes may have high malignant potential. showing a tumor.

(3) Confirmation of genetic abnormality within the gene sequence related to nm23-Hl or Hl . Nucleotide sequence analysis reveals nm23-Hl or Hl residues in tumor samples. It can be used to investigate gene structure. nm23-Hl and Hl nuclei The octide sequence defines the normal sequence.

These sequence changes in a patient's DNA indicate a tumor with high metastatic potential.

Human nm23DNA is suitable for producing human nm2B protein. It may also be incorporated into an expression vehicle.

Appropriate NA sequences can be included in any of a wide variety of vectors or plasmids. Good too. Such vectors include chromosomal, non-chromosomal and synthetic NA sequences; e.g. For example, derivatives of SV40: bacterial plasmids, phage DNA.

Yeast plasmid; plasmid and phage DNA, vaccinia, adenovirus derived from a combination of viral DNA such as , fowlpox virus, pseudorabies, etc. There is a vector.

Appropriate NA sequences may be inserted into the vector in a variety of ways. Generally, DNA Sequences are inserted into appropriate restriction endonuclease sites by techniques known in the art. Ru.

Such operations and others are believed to be within the knowledge of those skilled in the art.

The DNA sequence in the vector is placed in the appropriate expression vector to direct mRNA synthesis. operably linked to a troll sequence (promoter). promoters like this Typical examples include LTR or SV40 promoter, E. coli lac or trp , phage lambda PL promoter and other prokaryotic and eukaryotic cells or their Promoter known to control gene expression in viruses can be mentioned. The expression vector contains a liposome binding site for translation initiation and termination. Including rank. The vector may also contain sequences suitable for amplifying expression.

In addition, the expression vector can contain dihydrofolate reductase or Neomycin resistance or tetracycline or ampicillin resistance in E. coli Containing genes that provide phenotypic traits for selection of transformed host cells such as sex is preferable.

With the appropriate NA sequences and appropriate promoter or control sequences as described above. The containing vector is used to transform a suitable host and cause the host to express the protein. It may be used for Representative examples of suitable hosts include Escherichia coli, Salmonella typhimurium Bacterial cells such as Salwonella typhia + urius , fungal cells such as yeast, CHO or Bowes melanoma Examples include eel animal cells; plant cells. Selection of an appropriate host will be apparent to those skilled in the art from this disclosure. seems to fall within the range of

By conventional peptide chemistry, e.g. the use of peptide synthesizers and solid phase techniques It is also possible to produce human nm23 protein.

Human nm23 protein can be used to produce nm23 antibodies.

Antibodies against human nm23 protein are produced by procedures commonly known in the art. It's fine. For example, polyclonal antibodies can be used alone or coupled to appropriate proteins. It may also be produced by injecting the ringed protein into a non-human animal. appropriate After the period, the animals are bled and the serum is collected and purified by techniques known in the art. be done. Monoclonal antibodies can be used, for example, to target immune B-lymphocytes and myeloma cells. It is manufactured by the Kohler-Millstein (2) technique consisting of fusion.

For example, antigens such as those described above can be used to detect polyclonal antibody responses in mouse serum. Mice can be injected as described above until the time is reached. Mice were boosted again. Often, the spleen is removed and fusion with the myeloma is performed according to various methods. . Individual viable hybridoma cells first develop their ability to bind immunizing antigens. Therefore, their ability to immunoprecipitate nm23-Hl and Hl from cells tested for secretion of anti-nm23 antibodies. For this reason, human nm2 Antibodies induced in response to 3 proteins can be polyclonal or monoclonal antibodies. It can be any part of the body.

nm23 antibodies are associated with low levels of nm23 protein and thus metastatic or malignant It can be used to detect tumors with high potency. Such antibodies May be purified or not. Format for such assays (1) Immunohistochemical analysis, tumor sections were treated with anti-nm23-Hl Alternatively, the immune complex can be reacted with a peroxidase-labeled secondary antibody. Detected by standard and commercially available products such as antibodies. The density of such immunostaining From this, the amount of nm23-Hl or Hl produced in the cells can be evaluated.

(2) solid-phase immunoassays; such assays are performed in soluble extracts of tumor tissue; It can be used to quantitatively measure the amount of nm23-Hl and Hl in the sample.

In such assays, one component, either antibody or antigen, is attached to a solid support tumor. Fixed.

Therefore, according to another aspect of the invention, For detection or measurement of human nm23 protein using the above type of nm23 antibody assays are provided.

The assay technique used is that human nm23 antibody is present in the sample. It is supported on a solid support as a binding agent that binds to a protein, and the binding protein is Preferably, the assay is determined by the use of a specific tracer.

A tracer consists of a ligand labeled with a detectable label. That Riggan The code is immunologically linked to human nm23 protein, and Gunds may be labeled by techniques known in the art.

For this, for example, a human nm23 protein coupled to a nm23 antibody on a solid support may be used. Protein is measured by the use of nm23 antibody labeled with an appropriate detectable label. .

In such a sandwich assay technique, the labeled nm23 antibody is The antibodies may be clonal or polyclonal; e.g. The null antibody may be an antibody specific to human nm23 protein, and the antibody may be Procedures known in the art, such as inoculating suitable animals with human nm23 protein. It may be produced depending on the crop.

Detectable labels include enzymes, radiolabels, total chromogens (both fluorescent and/or adsorbed dyes). Any of a variety of detectable age groups, including (including), etc. detectable The choice of label will be within the skill in the art from this disclosure.

The solid support for the nm23 antibody is a variety of solid supports.

Selection of an appropriate support will be within the scope of those skilled in the art from this disclosure. It seems to belong to For example, solid supports include microtitration plates, tubes, particles, etc. However, the scope of the present invention is not limited to any representative support. stomach. The nm23 antibody can be prepared using techniques known in the art, e.g. coating, covalent coupling. It is supported by a support body by a ring or the like. Selection of appropriate techniques will be within the skill of those skilled in the art from this disclosure. It seems to belong within the range.

Sandwich assays can be performed using various techniques, e.g. ), “reverse” or “simultaneously.”

However, forward techniques are preferred.

In a typical operation, the nm23 antibody supported on a solid support is specifically with such antibodies on the support any of these proteins present. In order to bind, first, a protein containing human nm23 protein or suspected to contain human nm23 protein is prepared. contact with the sample.

After washing the solid support, the support is coated with a tracer that binds to human nm23 protein. - be contacted. If such proteins are present in the sample, the tracer becomes bound to such proteins bound to antibodies on a solid support, The presence of tracer on the solid support and the presence of human nm23 in the sample Indicates the presence of protein. The presence of a tracer can be determined by procedures known in the art. Although the ζ'λ operation is a switch assay, the nm et al. 3 antibody is compatible with other assay techniques. techniques, e.g. aggregation where the nm23 antibody is used on one solid particle, such as a latex particle. It is believed that it can be used in assays.

According to another aspect of the invention, nm23 antibodies, preferably nm23 proteins Measurement of human nm23 protein containing nm23 antibodies induced in response to The assay kit package is provided for you. nm23 antibody is a detectable antibody. May or may not be marked with a marker or sign. The kit performs immunohistochemical assay. When used for It may also contain a labeled antibody that binds immunologically.

If the kit is used in an immunoassay, the kit will contain supporting nm23 antibodies and and unsupported nm23 antibody, which may contain a detectable label or Preferably, it is labeled with a marker. The kit contains other materials such as buffers etc. It may also contain ingredients.

The invention is further illustrated in the examples below; however, the scope of the invention extends thereto. less restricted. In the examples, unless otherwise indicated, purification, cleavage and ligation are If +Mo1ecular Cloning, a1abo’ratory anua+- (molecular cloning, experiment manual) by Maniatis et al, coldSpring Harbor Laboratory ( (1982').

Example 1 Two separate cDNAs were prepared from normal human fibroblast mRNA. isolated from a library. Standard techniques were used throughout. as a probe , we used the 502-group Hpall restriction fragment of pnm23-M1 . Ste'eg et al. (3). This DNA was transferred to an agarose gel using a DE45 membrane. Isolated from pneumophoresis (Schlicherand et al.) DNA was subjected to nick translation reaction (Asersham kit) and p3 Activation was performed using 2PdCTP (Avtersham). Old roto Oka cDNA live obtained from yala (Okayama et at, (4)) Individual bacteria of the rally were dispersed onto agaro-surlia broth plates. After proliferation Transfer them to nitrocellulose for 5chlicher and 5chlicher. ell), 0. Eluted with 5M NaOH and 1.5M NaCl and neutralized with IMNHAc. Fix DNA to nitrocellulose by baking Ta. Hybridization with radioactive probe in 40% formamide, 0.75M NaC1, 0,075M Na citrate, 0.2% bovine serum albumin, 0.2% Fluid Ficol sO, 2% polyvinylpyrrolidone and 2 sg/m It was carried out in 1DNA. Hybridization was performed at 42°C for 15 hours. Yes After bridging, the filter was soaked in 0.3M NaCl 1° for 20 minutes at room temperature. Washed twice with 0.03M Na citrate, then incubated at 42°C for 20 min each. ．． Washed twice with 015M NaCl and o,oox 5M Na citrate. positive c Ibrid formation was detected by autoradiography for five bacteria. this were purified by single cell cloning.

DNA was extracted from each of the five clones and analyzed by restriction enzyme analysis. separate putter The pattern was confirmed for two clones, pnm23-Hl and pnm23-H2. child These were subjected to the following analysis.

The DNA sequences of pnm23-Hl and pnm23-H2 are dideoxy chainer Mination method (5) (U, S.

It was determined using BIOehe layer 1cal kit). For this purpose, pn The Xhol fragments of m23-Hl and pnm23-H2 were removed from the plasmid and the It was inserted into the 5alI site of M13mp18 (BRL) using standard techniques.

DNA sequence analysis uses synthetic 17 base oligonucleotides as reaction primers. I went there. The DNA sequences of pnm23-Hl and pnm23-H2 are shown in Figure 2 and and Fig. 3. Figure 2 shows that pnm23-Hl is located at the putative translation start codon ( nucleotide sequence upstream of nucleotide 87). Figure 3 pnm23-H2 makes a partial copy of the mRNA starting in the open reading frame. Indicates that it contains. The non-identity of the nucleotide sequence (similarity rate 94%) is pnm23 -Hl and pnm23-H2 are products of different genes.

Example 2 - Production of nm23-Hl and nm23-H2 proteins The nucleotide sequences of pnm23-H1 and H2 are related to the corresponding proteins. can be translated into the predicted protein sequence. There are several ways like this Can be used to form proteins.

(1) Standard chemical operations include nm23-Hl or ■ all or part of the 2 amino acid sequence. can be used to synthesize peptides corresponding to . These peptides are Can be coupled to carrier proteins such as KLH for antibody production can.

(2) Corresponds to all or a part of nm23-Hl or a part of nm23-H2 Proteins can be synthesized within bacteria under the direction of bacterial transcriptional promoting signals. n The m23-Hl protein was expressed in a vector similar to that described in (6). It is expressed under the direction of the bacteriophage lambda PL promoter. child The vector was constructed as shown in FIG. Plasmid pBR322 Digested with coRI and Aual, and the base fragments were isolated by agarose gel electrophoresis. . This includes several enzyme sites, the bacterial liposome binding site and the Ncol restriction enzyme site. A synthetic restriction fragment (FIG. 1) containing a translation initiation codon containing a position-containing translation initiation codon was combined. T these 4, react with DNA ligase, integrate into E. coli, and transform to obtain a protein with the correct structure. Confirmed lasmid. DNA from these plasmids was combined with BstXI and Bam 4. Cut the bacteriophage DNA with HI. 5kbHi nd III Combined with BstXI-Bgll+ cleavage of the fragment. This BstXI-Bgllll The fragment contains the PL promoter. binding and E. coli (containing cI857 brophage) After transformation with the following plasmids, a plasmid containing the structure shown in FIG. 1 was confirmed. this Cut the DNA from the plasmid with BstXI and Hpal, and add each DNA. The sticky ends were supplemented with E. coli DNA polymerase engineering fragment. Conclude this under standard conditions. The cells were combined, integrated into E. coli, and transformed (7). nm2-Hl is supercoil to produce partially truncated molecules, as seen in the conversion of conjugated molecules to linear forms. Cut with Ncal at a ratio of 1 unit of enzyme per 1 μg of DNA for 2 minutes. It was removed from M13mp18. This was extracted with phenol/CHC12. and ethanol precipitation to remove the NcoI enzyme, and further cleavage with EcoRI. . A 0.7 kb fragment was isolated from agarose gel electrophoresis. EcoR this fragment It was mixed with plasmid pPL cut with I and NcoI, ligated, and assembled into E. coli. and transformed.

A bacterial clone capable of directing the synthesis of human nm23-Hl protein was identified.

Bacteria were grown at 32°C until it reached 0D660-1, and the temperature and degree were shifted to 42°C. Grow for 16 hours at °C. Total bacterial proteins were added to 15% polyacrylic with SDS. Tested by electrophoresis in containing amide gels. human nm23-Hltan 1 anti-peptide antiserum against amino acids 86-102 of the protein. It was confirmed as a 19 kDa protein that can react with

Human nm23-Hl protein can be purified from bacteria by various methods. example For example, after growth and temperature shift induction, the bacteria were incubated with 20mM) at pH 7.5, 150n Eluted with sonication in MNaCl (TBS). 100. The cells were removed by centrifugation at 000Xg for 30 minutes. Then add 60% ammonium sulfate A saturated solution was added and the proteins were allowed to precipitate for 10 minutes at 4°C. These types Proteins were collected by centrifugation at 100,000xg for 10 minutes, and the precipitate was dissolved in TBS. Dissolved. After 716 hours of dialysis, remove the fine sediment at 10,000×g for 10 minutes. Collect by centrifugation. It is solubilized in TBS and 1 mM TT. like this The protein obtained was determined by SDS polyacrylamide gel electrophoresis. Roller purity is 80% or more. The proteins thus obtained are useful for antibody production and biology. It is suitable for use as an immunizing antigen in functional modification experiments.

The nucleotide sequences of nm23-Hl and H2 are similar to those of either protein in eukaryotic cells. express quality. Proteins in cells ranging from yeast to human tissue culture cells There are various systems available for the expression of . nm23-Hl or H2 protein The essential elements required for the expression of nm23 are nucleotide sequences capable of directing the synthesis of nm23. there were.

Example 3 - Production of nm23 antibody The products described in the Example 3 section can be used as antigens. These are as they are Until or Keyhole Limpid hemocyanin It can be used by coupling to a carrier protein such as. cupri This can be done using established techniques and using crosslinking agents such as EDC. next Mix the antigen with an adjuvant (e.g. Freund's) (rabbit, rat or inject into animals (such as goats). with an adjuvant (e.g. Freund's incomplete) After a booster injection with mixed antigen, animals are bled and serum is prepared. antibody The presence of ISA).

Polyclonal antisera against nm23-Hl or H2 were prepared using affinity chromatography. It can be obtained in purified form by tography. Immunoglobulin molecules are It can be obtained from serum by binding to Daphylococcal protein A, and anti-nm23-H1 or H2 is linked to a solid matrix on which the appropriate nm23 antigen is chemically immobilized. I got more.

Example 4 - Production of monoclonal antibodies Ba l b/c mice were immunized with complete Freund's adjuvant and Purified nm23-Hlta mixed with incomplete Freund's adjuvant for boosting. Immunizations were made with 3 IP injections of 100 μg of protein at 1 week intervals. hybrid The author is Lane et al., Methods in Enz, 121. p .

Obtained by the method of [13 (198B). The fusion partner is myeloma P3x63-A It was Ag8.653 obtained from TCC. Lane et al. ,, Hybridosa, Vol. 7. Obtained by the method of p. 289 (198 g) Intraperitoneal cells were added. l-ibridoma by NCTC-109 (Gibco) ), 7. 5% tallow baby serum (Sigma), 7.5% CS P R-3 ( Sigma), 1mM Na pyruvate, 100 units penicillin, 100μg Treptomycin, 10μg/ml insulin and 25μNβ-mercaptoe Supplemented with ethanol, 0.1 μM ahyboxanthin, 0.4 μM amphobuterin and Grown in DMEM containing 0.016mM thymidine. hybridomac Roans were grown in 96-well dishes for two weeks. Anti-nm23 producing hybridoma was confirmed by ELISA. The purified nm23IH protein was purified by IMURON (1 +1llulon) in one dish and incubate the noibridoma medium at room temperature for 2 hours. I responded.

The antibody reaction was carried out using the BRL HYBRL kit under the following conditions: biotinylated goat anti-mouse anti-antibody body and stevtavicudin (steptavfcdfn) horseradish peroxida Detection was performed using For positive/1 hybridomas, 5% hybridoma growth aid ( The clones were cloned by limiting dilution in the above-mentioned medium containing J. Fisher. this were tested for reactivity by ELISA. The reaction with human nm23 protein is Confirmed by Western blotting assay.

Example 5 - Detection of metastatic tumors Antibodies specific for human nm23-Hl and H2 proteins are obtained as described above. be able to. nm23- One this for amino acids 45-61 of the H1 sequence It was used like this. Standard techniques can be used to prepare sections for immunohistochemistry. can. These methods include cryosectioning or formalin fixation of the sample. There is post-paraffin embedding. In this example, tumor sections were prepared in 10% neutralized buffered forma. Fixed overnight in phosphorus and processed using an automated tissue processor (Fisher). It was embedded in Ruffin.

Cut 5 micron sections and use standard procedures requiring xylene and alcohol. Deparaffinized. The sections were then incubated 1/2 with an affinity-purified anti-peptide antibody. Immunostaining was carried out at a dilution of 200: Immunostaining was carried out using a biotinylated goat anti-rabbit antibody. using avidin biotinylated horseradish peroxidase (Vect It was performed by standard techniques as described by or). 0 for 5 minutes at room temperature , after color reaction (diaminobenzidine tetrahydrochloride) at 5 gg/l , and washed with water to stop the reaction.

Sections were then counterstained with Mayers hematoxylin and Dehydrated and coverslips applied using standard methods. Sections are then stained with clear cytoplasm Therefore, it was examined using an optical microscope. Two patients with breast cancer whose tumor had spread to the axillary lymph nodes Samples show little staining; 2 samples from patients whose cancer was confined to the chest show different staining.

This is because detection of low nm23 protein expression tends to spread beyond the initial site of malignancy. Indicates that a tumor has been confirmed.

Various modifications and variations of the present invention are possible in light of the above disclosure; therefore, the appended Within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

References 1, Lehraeh, H., Blocheslstry, 1B, p. 4743 (1975).

2, Kohler - Old 11stein, Ga1f're, G, and old 1st eln,c,.

Methods Enz, 73. p, 1 (1981).

3. Steeg et al., JNCl, 80. p, 200 (1988) .

4, Okayawa, H. and Berg, P., Mo1. Ce1l Bio l,,3. p., 280 (1983).

5. Sanger, P. et al., Proc. Nat'l AcadJc i, USA, 74°9.5463 (1977).

6. Rosenberg, M., and 5hatZlan A., Method. ds Enz, 101.1), 123 (1983).

7. Transformation 1nto E. coli (E. coli) Transformation), Maniatis, T. et al., Molecular Cl oning, ColdSpring Harbor Laboratory (1 982).

Explanation of Figure 1 A-Aval; R-EcoRI; B-BamHI; Bx=Bs tXI ;H-Hpa I;N-Nco I, bacteriophage lambda PL promoter The target position is indicated by an arrow and "PL". lambda bacteriophage lambda N Genes are indicated by boxes and "N genes". The position of the liposome binding sequence is S1 Indicated by 0. The position of the protein initiator atg is indicated by 'atg'. It will be done. A binding reaction occurs at the point where the two arrows come together.

pPL-nm23-HI FIG.1 TGCTGCGAA CCA CGT GGG TCCCGG GCG CGT TTCGGG TGCTGG CGG CTGGAG TTCTCCCTG TACAGT (iTT ACCA Ding CCCC GACCAT CTG ATT AAA ATGCTT CCT CCC pO1yA FIG, 2AGA G GCAAACAGG An GAT CAT TCT TTT ATA GAG CAT　ATT　TGCCAATAA　AGCTTT　TGG　AAG　CCG (poly A) FIG, 3 international search report

Claims (21)

[Claims] 1. Human nm23 DNA. 2. 2. The human nm23 DNA of claim 1, comprising the sequence of FIG. 3. The human nm23 DNA according to claim 1, comprising the sequence of FIG. 4. nm23 antibody. 5. Human nm23 protein. 6. 6. The human nm23 protein according to claim 5, having the sequence of FIG. 7. 6. The human nm23 protein according to claim 5, having the sequence of FIG. 8. A sample obtained from a human is brought into contact with nm23 antibody, and the nm23 antibody and determining the presence of human nm23 in a bound sample. 3 protein analysis method. 9. An expression vehicle comprising the DNA according to claim 1. 10. A host genetically engineered with the DNA according to claim 1. 11. 5. The antibody of claim 4, which is induced in response to human nm23 protein. 12. A host genetically engineered with the DNA according to claim 2. 13. A host genetically engineered with the DNA according to claim 3. 14. An antibody that recognizes the protein according to claim 6. 15. An antibody that recognizes the protein according to claim 7. 16. 5. The antibody according to claim 4, wherein the antibody is a monoclonal antibody. 17. A method for predicting the malignant potential of human tumors, the method comprising: To predict the malignant potential of human tumors, the immunoreaction with nm23 antibody A method comprising detecting human nm23 protein. 18. nm23 antibody was induced in response to human nm23 protein 18. The method of claim 17. 19. 19. The method of claim 18, wherein the antibody is a monoclonal antibody. 20. 2. The DNA according to claim 1, wherein the DNA encodes a full length human nm23 protein. DNA. 21. 6. The protein according to claim 5, wherein the protein is full chain length human nm23 protein. protein.